Key points are not available for this paper at this time.
c-Jun NH2-terminal protein kinase (JNK), a distant member of the mitogen-activated protein (MAP) kinase family, regulates gene expression in response to various extracellular stimuli. JNK is activated by JNK-activating kinase 1 (JNKK1), a dual specificity protein kinase that phosphorylates JNK on threonine 183 and tyrosine 185 residues. Here we show that JNKK2, a novel member of the MAP kinase kinase family, was phosphorylated and activated by MEKK1, a MAP kinase kinase kinase in the JNK signaling cascade. JNKK2 activity was also stimulated by constitutively active forms of Rac and Cdc42Hs, members of the Rho small GTP-binding protein family. Unlike JNKK1 that activates both JNK and p38 MAP kinases, JNKK2 stimulated only JNK. Transient transfection assays demonstrated that JNKK2 potentiated the stimulation of c-Jun transcriptional activity by MEKK1. The existence of multiple JNK-activating kinases may contribute to the specificity of the JNK signaling cascade. c-Jun NH2-terminal protein kinase (JNK), a distant member of the mitogen-activated protein (MAP) kinase family, regulates gene expression in response to various extracellular stimuli. JNK is activated by JNK-activating kinase 1 (JNKK1), a dual specificity protein kinase that phosphorylates JNK on threonine 183 and tyrosine 185 residues. Here we show that JNKK2, a novel member of the MAP kinase kinase family, was phosphorylated and activated by MEKK1, a MAP kinase kinase kinase in the JNK signaling cascade. JNKK2 activity was also stimulated by constitutively active forms of Rac and Cdc42Hs, members of the Rho small GTP-binding protein family. Unlike JNKK1 that activates both JNK and p38 MAP kinases, JNKK2 stimulated only JNK. Transient transfection assays demonstrated that JNKK2 potentiated the stimulation of c-Jun transcriptional activity by MEKK1. The existence of multiple JNK-activating kinases may contribute to the specificity of the JNK signaling cascade. The mitogen-activated protein (MAP) 1The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-related kinase; JNK, c-Jun NH2-terminal protein kinase; JNKK, JNK-activating kinase; MEKK, MAP kinase kinase kinase; ATF, activating transcription factor; HA, hemagglutinin; CBSP, CSAID™-binding protein. kinase is an essential part of the signal transduction machinery and occupies a central position in cell growth, differentiation, and transformation (1Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2258) Google Scholar, 2Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar, 3Hunter T. Cell. 1997; 88: 333-346Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). 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Science. 1993; 260: 315-319Crossref PubMed Scopus (875) Google Scholar). In the JNK signaling cascade, the MAP kinase kinase is JNK-activating kinase (JNKK) 1 (also known as SAPK/ERK kinase, SEK, and MKK4) (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar, 21Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (917) Google Scholar, 22Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar), and the MAP kinase kinase kinases are the MEKKs (23Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar, 24Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. 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The MAP kinase kinase kinases for p38 may include MEKK1, TAK1, and Ask (28English J.M. Vanderbilt C.A. Xu S. Marcus S. Cobb M.H. J. Biol. Chem. 1995; 270: 28897-28902Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 29Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar, 30Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2037) Google Scholar). These individual MAP kinase modules may provide a structural basis for different signaling cascades to relay extracellular stimuli to specific effectors. A challenge in understanding the mammalian MAP kinase cascade is how signaling specificity is achieved. Despite the MAP kinase modules, cross-talk still exists. The cross-talk may allow the cells to coordinate the activity of different signaling cascades to produce a specific physiological response. However, it also makes the signaling cascades prone to lack of specificity. Other mechanisms are needed to ensure the specificity of each MAP kinase cascade, including subcellular localization, specific associated proteins, high enzymatic specificity, and selective responsiveness to extracellular stimuli. The JNK cascade is activated by various stimuli such as growth factors, cytokines, tumor promoters, protein synthesis inhibitors, ultraviolet (UV) light irradiation, and oncogenes (1Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2258) Google Scholar). JNK in turn stimulates the activity of several transcription factors including c-Jun, ATF-2, Elk, and Sap-1 (6Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1710) Google Scholar, 7Derijard B. Hibi M. Wu I.H. Barret T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 8Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar, 31Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1339) Google Scholar, 32Whitmarsh A.J. Shore P. Sharrocks A.D. Davis R.J. Science. 1995; 269: 403-407Crossref PubMed Scopus (882) Google Scholar, 33Cavigelli M. Dolfi F. Claret F-X. Karin M. EMBO J. 1995; 14: 5957-5964Crossref PubMed Scopus (489) Google Scholar). It is not completely understood how the specificity of the JNK cascade is maintained. One plausible mechanism is through the existence of multiple JNKKs that have high substrate specificity and respond to distinct upstream signals. To test this hypothesis, we have isolated a novel JNK-activating kinase, JNKK2, which is a close homologue of JNKK1. In contrast to JNKK1, which is selectively expressed in skeletal muscle and brain, JNKK2 is expressed in many tissues examined. Unlike JNKK1 which activates both JNK and p38, JNKK2 stimulated only JNK. Furthermore, JNKK2 did not respond to several extracellular stimuli that activate JNKK1. These data suggest that JNKK2 is a specific JNK activator and may contribute to the specificity of the JNK cascade. HeLa cells were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. A human JNKK1 cDNA (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar) was used as a probe to isolate JNKK1 homologues by screening a λZAPII HeLa cDNA library (Stratagene) at low stringency. Thirty positive clones were obtained after screening 2 × 106 phage. These clones were grouped and sequenced on both strands by the dideoxy chain termination method using Sequenase Version II (U. S. Biochemical Corp.). Nucleotide sequence comparison of clones 4 and 23 with the GenBankTM data base reveals five sets of nucleotide sequences (GenBankTM accession numbers AA194047, AA019720,AA194193, AA258025, and AA252650) that are nearly identical. These clones were partially sequenced by the Washington University-Merck EST project. The clones AA194047 and AA019720 were requested and completely sequenced. The JNKK2 expression vector was constructed by inserting a polymerase chain reaction-generatedNcoI-BglII fragment encoding JNKK2 intoNcoI and BglII sites of pSRα3-hemagglutinin (HA) vector (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar). To construct pGEX-KG-JNKK2, the BglII site of the NcoI-BglII fragment of JNKK2 was blunted, and the fragment was inserted between the NcoI site and blunted HindII site of pGEX-KG vector. A Chameleon™ mutagenesis kit (Stratagene) was used to replace Ser-272 and Thr-276 with alanines, to create pGEX-KG-JNKK2 (AA). The expression vectors of MEKK1, Rac1, Rac2, Cdc42Hs, RhoA, JNK1, p38, MKK6, MEK1, ERK2, c-Jun, and ATF2 have been described (6Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1710) Google Scholar, 20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar, 23Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar, 26Han J. Lee J-D. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 34Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar, 35Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Wound G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1263) Google Scholar). GST-JNKK2, GST-JNKK(AA), GST-JNK1, GST-p38, GST-c-Jun, and GST-ATF2 were purified on glutathione-agarose, as described (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar). Histidine-tagged ERK2 was purified by a nickel-chelate column, according to the manufacturer's procedure (Pharmacia Biotech Inc.). HeLa cells were transiently transfected with expression vectors and harvested as described (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar). HA-tagged or M2-Flag-tagged protein kinases were immunoprecipitated with specific antibodies for 3 h at 4 °C. The activity of the immune complex was assayed at 30 °C for 30 min in 30 μl of kinase buffer (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar) in the presence of 10 μm γ-32PATP (10 Ci/mmol) with appropriate substrates, as indicated in the figure legends. The proteins were resolved by 13% SDS-polyacrylamide gel electrophoresis, followed by autoradiography. The phosphorylated proteins were quantitated by a phosphorImager. Sequence analysis revealed that the JNKK2 clone encodes a complete open reading frame which appears to be a novel MAP kinase kinase (Fig. 1). The new MAP kinase kinase is closest to hep, aDrosophila JNK-activating kinase (36Glise B. Bourbon H. Noselli S. Cell. 1995; 83: 451-461Abstract Full Text PDF PubMed Scopus (288) Google Scholar) (56.2% identity), followed by human JNKK1 (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar) (42.4% identity), based on the comparison of all known MAP kinase kinases (PILE-UP program, Wisconsin Genetics Computer Group). Northern blot analysis revealed that unlike JNKK1, which is expressed mainly in skeletal muscle and brain, JNKK-2 is widely expressed in many tissues, with the highest level of expression in skeletal muscle (data not shown), suggesting that the two JNK activators may have different roles in different cells. We and others have shown that MEKK1 acts as a MAP kinase kinase kinase which directly phosphorylates and activates JNKK1 (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar, 23Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar, 24Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (660) Google Scholar). In addition, Rac and Cdc42Hs, members of the Rho small GTP-binding protein family, are both strong activators of JNKK1 and JNK (34Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar, 37Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar, 38Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1059) Google Scholar). The effects of expression of MEKK1 and Rac/Cdc42Hs on JNKK2 activity were examined in transient transfection assays. Coexpression of the active forms of MEKK1, Rac1 and -2, and Cdc42Hs strongly activated JNKK2 (Fig.2 A). The active form of RhoA did not activate JNKK2 (Fig. 2 A) but was able to stimulate ERK activity (data not shown). MEKK1 also directly phosphorylated JNKK2 but not the JNKK2 (S272A/T276A) mutant, in which the putative activating phosphorylation residues Ser-272 and Thr-276 were replaced with alanines (Fig. 2 B). These results demonstrate that JNKK2 is a JNK-activating kinase which acts downstream of MEKK1 and Rac/Cdc42Hs in the JNK signaling pathway. We also examined the regulation of JNKK2 by UV and the protein synthesis inhibitor anisomycin, both of which are strong activators of JNKK1 (Fig. 2 C, top panel). UV and anisomycin only weakly stimulated JNKK2 activity (Fig. 2 C, middle panel), even though JNK activity was stimulated manyfold in the same transfection assays (Fig. 2 C, bottom panel). It is likely that JNKK2 may respond to upstream signals other than UV and anisomycin. MEKK1-activated HA-JNKK2 was isolated from transfected HeLa cells, and its substrate specificity was determined by protein kinase assays. JNKK2 efficiently phosphorylated GST-JNK1, but not GST-p38 or histidine-ERK2 (Fig.3 A). However, GST-p38 and histidine-ERK2 can be phosphorylated by their specific upstream kinases, MKK6 and MEK1(EE), respectively (Fig. 3 A). The phosphorylation of JNK by JNKK2 is specific, since JNKK2 did not phosphorylate mutants JNK1 (APY) or JNK1 (APF), in which the activating phosphorylation residues Thr-183 and Tyr-185 were replaced by alanine and phenylalanine (Fig. 3 B). Phosphorylation of JNK by JNKK2in vitro led to JNK activation, as measured in a coupled kinase assay (Fig. 3 C). The effect of JNKK2 on JNK in vivo was examined in transient transfection assays. In HeLa cells, cotransfection of JNKK2 stimulated M2 Flag-tagged JNK1 (M2-JNK1) activity (Fig. 3 D). The stimulation by JNKK2 was further potentiated by a suboptimal amount of cotransfected MEKK1 (Fig. 3 D). Under the same conditions, JNKK2 or JNKK2 plus MEKK1 did not stimulate activity (Fig.3 D). These data demonstrate that JNKK2 is a specific activator of JNK in To the effect of JNKK2 on c-Jun transcriptional HeLa cells were cotransfected with the active form of MEKK1 and protein (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar). MEKK1 stimulated activity as measured by a gene by a Coexpression of JNKK2 potentiated the effect of MEKK1 on in (Fig. MEKK1 MEKK1 plus JNKK2 stimulated the activity of in which both and have been replaced with alanines (Fig. These results demonstrate that JNKK2 in stimulation of c-Jun transcription The specificity of the MAP kinase cascade is in by enzymatic specificity of MAP kinase kinases in the MAP kinase module M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar). JNKK1 was shown to be an MAP kinase kinase of vitro (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar, 21Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (917) Google Scholar, 22Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar) and is for JNK vivo H. L. A. R. Rubie E.A. A. T.W. Woodgett J.R. J.M. Nature. 1997; PubMed Scopus Google Scholar, D. C. M. Xu J. Davis R.J. S. A. 1997; PubMed Scopus Google Scholar). However, JNKK1 is also in regulation of p38 (20Lin A. Minden A,. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar, 22Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar). In contrast to JNKK1, JNKK2 is a selective kinase that phosphorylates and activates JNK but not p38 or ERK2 (Fig. 3 A). The high substrate specificity of JNKK2 contribute to the specificity of the JNK cascade. The specificity of the JNK cascade may also be by the responsiveness of JNKKs to a of upstream stimuli. It been shown that various extracellular stimuli and stimulate JNK of which through activating JNKK1. JNKK2 can be activated by MEKK1, and Cdc42Hs, which are activators of JNKK1 (Fig. 2 A). However, JNKK2 was only weakly activated by UV and anisomycin (Fig. 2 both of which strongly activate JNKK1 (Fig. 2 C). stimulates JNKK1, but only weakly stimulated JNKK2 activity (data not shown). The response of JNKK1 and JNKK2 to extracellular stimuli that signaling to JNK may upstream of JNK at the level of It is that JNKK2 may the of JNK by extracellular stimuli that stimulate only JNK but not of extracellular stimuli that activate JNKK2 provide the specificity of the JNK signaling pathway. We A. Minden and J. for and and M. A. M. G. N. and J. Han for the different that this We also members of and for reading the
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